aguA , the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis , Is Specifically Induced on Xylose and Not on Glucuronic Acid

Abstract

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression ofaguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

Biochemical characterization and identification of catalytic residues in α‐glucuronidase from Bacillus stearothermophilus T‐6

“…As with other bacterial α‐glucuronidase sequences, the aguA gene does not include any recognizable Gram‐positive bacterial signal peptide sequence [30], thus α‐glucuronidase T‐6 is intracellular. The fungal α‐glucuronidases from T. reesei and A. tubingensis have a signal peptide of 19 and 20 amino acids, respectively, and appear to be extracellular [10,12]. These fungal α‐glucuronidases are about 150 amino acids longer (at the C‐terminus) than the bacterial α‐glucuronidases, and this additional region may be specific for fungal α‐glucuronidases [12].…”

“…About 15 α‐glucuronidases from various fungi and bacteria have been purified and biochemically characterized. To date, six α‐glucuronidase genes have been cloned and sequenced, and the purification of the gene products of four of them have been reported [10–13]. Based on amino‐acid sequence similarities, α‐glucuronidases have been classified to family 67 in the general classification of the glycosyl hydrolases [14,15].…”

Xylanase XYN IV from Trichoderma reesei showing exo‐ and endo‐xylanase activity

“…HexA 3 Xyl 3 is a substrate for xylanases that catalyze cleavage from the reducing end Here we show that the non-natural hexenuronic acidcontaining xylooligosaccharide, tetrasaccharide Hex-A 3 Xyl 3 , may serve as a specific substrate for screening of exoxylanases operating on the reducing end because this oligosaccharide is not attacked by a-glucuronidases. In contrast, the natural oligosaccharide MeGlcA 3 Xyl 3 is attacked by a-glucuronidases [33,[35][36][37] and thus is not suitable for screening of exoxylanases in the presence of a-glucuronidases. In the present work, use of HexA 3 Xyl 3 enabled discovery of a new type of xylanase from T. reesei RUT-C30.…”

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger

“…MMXA cultures were grown for three days, after which conidia were harvested using Saline-Tween (0.8% NaCl and 0.005% Tween-80). 250 ml liquid cultures were inoculated with 1.25*10 9 . This formula was inferred from the TOF of beads with diameters of 42 µm, 250 µm and 500 µm.…”